Ultra-Processed Foods and Schooling Are Independently Associated with Lower Iron and Folate Consumption by Pregnant Women Followed in Primary Health Care

Combined deficiencies of nutrients such as iron and folic acid intake during pregnancy are related to nutritional deficiencies risk, such as anemia. The objective of this study was to analyze the association between risk factors (sociodemographic, dietary and lifestyle) and the intake of iron and folate by pregnant women followed up in Primary Health Care (PHC) in the Federal District, Brazil. A cross-sectional observational study was carried out with adult pregnant women of different gestational ages. A semi-structured questionnaire was applied by researchers trained to collect sociodemographic, economic, environmental, and health data. Two nonconsecutive 24-hour recalls (24hr) were carried out to collect data about food consumption. Multivariate linear regression models were used to analyze the association between sociodemographic and dietary risk factors and the consumption of iron and folate. The mean daily energy intake was 1726 kcal (95% CI 1641–1811), with 22.4% (95% CI 20.09–24.66) derived from ultra-processed foods (UPFs). The mean iron and folate intake were 5.28 mg (95% CI 5.09–5.48) and 193.42 µg (95% CI 182.22–204.61), respectively. According to the multivariate model, the highest quintile of ultra-processed foods intake was associated with lower iron (β = −1.15; IC 95%: −1.74; 0.55; p < 0.001) and folate intake (β = −63.23; IC 95%: −98.32; −28.15; p < 0.001). Pregnant women with high school degree presented higher iron intake (β = 0.74; IC 95%: 0.20; 1.28; p = 0.007) and folate intake (β = 38.95; IC 95%: 6.96; 70.95; p = 0.017) compared to pregnant women with elementary school degree. Folate consumption was also associated with the second gestational period (β = 39.44; IC 95%: 5.58; 73.30; p = 0.023) and pregnancy planning (β = 26.88; IC 95%: 3.58; 50.18; p = 0.024). Further research is warranted to enhance evidence on the relationship between the role of processed foods and micronutrients intake to strengthen the nutritional quality of diet of pregnant women attended in Primary Health Care.


Introduction
Poor nutritional quality of the diet during pregnancy can harm mothers' health and the development of the fetus. To ensure healthy gestational development, it is important to adequate macro and micronutrient supply, such as carbohydrates, proteins, vitamins, and minerals in order to meet the nutritional needs of both mother and fetus [1,2]. In addition, during pregnancy, it is particularly relevant to consume a variety of fresh and minimally processed foods and water, to meet the need for essential nutrients for this life event, such as iron and folate [3]. study -EMDI Brasil" carried out in the Federal District, Brazil, between August 2019 and September 2021.
To perform this study a simple random sample was calculated using the StatCalc tool of the EpiInfo Software version 7.2 (Center for Disease Control and Prevention, Atlanta, GA, USA), considering the average monthly prenatal care appointments at PHC in 2016 as a proxy for the number of pregnant women monitored by PHC in Federal District (n= 18,877data reported by the Federal District State Department of Health). The prevalence of the indicator "consumption of ultra-processed foods the day before" among Brazilian pregnant women monitored by PHC (81.5%), in the same year, from the Food and Nutrition Surveillance System -SISVAN [28]. The acceptable error of 5% and the 95% confidence interval (95% CI) were considered. At the end, the minimum number of pregnant women to be included was defined as 190, and to anticipate possible sample losses, 10% was added to the estimated number. Thus, the sample was estimated at 209 pregnant women. Also, ten PHC units were selected according to the proximity to the central region and the highest monthly average prenatal care performed in 2016 (data reported by the Federal District State Department of Health), resulting in a representative sample of the studied population.

Inclusion and Exclusion Criteria
Pregnant women from all gestational age (first, second and third trimesters) who attended the selected PHC units for prenatal follow-up appointments were invited to participate at each unit. Pregnant women who were not able to answer the questionnaire or decline were excluded from the sample. Pregnant who had any physical changes that could distort the research results, such as a history of thyroid disease or surgery, a reported diagnosis of hypo or hyperthyroidism, were excluded from the study, as they could interfere with the primary outcomes of the EMDI multicenter study. Figure 1 details the eligibility process.

Data Collect
Data collection was conducted from August 2019 to September 2021, through faceto-face interview. A semi-structured questionnaire was applied by researchers trained to collect sociodemographic, economic, environmental and health data from pregnant women as follow: maternal age (in years), access to government social program (yes or no); self-reported skin color (white, brown/black, or asian); maternal education (no education, elementary school, high school, or higher education); household income in the previous month (up to USD 94.00; between USD 94.00 and USD 188.00; between USD 188.00 and USD 566.00; above USD 566.00 or not informed); use of iron supplementation (yes or no); use of folic acid supplementation (yes or no); use of multivitamins (yes or no); home-made meal frequency (in days); previous pregnancies (none, between 1 and 3, between 4 and 6, or more than 6) and planned pregnancy (yes or no). Information regarding anthropometry (pre-gestational weight, current weight, height), and obstetric history (date of last menstrual period, date of last ultrasound) were collected from the Pregnant Woman's Handbook. Nutritional status was classified according to WHO pre-pregnancy BMI values (<18.5 underweight, ≥18.5-24.9 normal weight, ≥25.0-29.9 overweight and ≥30.0 obese) [29].
The first 24-hour recall (24hr) was carried out to collect data on food consumption. A second 24hr was collected randomly by telephone in 20% of the sample up to 15 days after the first on non-consecutive days to correct the within-person variability [30,31].
During the interview to apply the 24hr, the US Department of Agriculture's 5-step multiple-pass method was used, according to the 5 steps established by the institution: (1) initial quick list of foods and beverages consumed; (2) list of commonly forgotten foods; (3) investigation of meal times and occasions; (4) detailing of previous information including description of quantity, method of preparation, additions and brands; and (5) final review of recall information [32]. To assist pregnant women in quantifying the portions consumed, the Photographic Manual of Food Quantification was used, which contains photos of portions, forms of food and household measures [33].
The food consumption data from 24hr were entered for nutrients composition using the Globodiet software, Brazilian version, Data Entry mode, developed by the GloboDiet Initiative [34] and analyzed for nutrient composition using the Brazilian Food Composition Table (TBCA) [35]. Analysis of food consumption included assessment of total energy intake, calories consumed from UPFs, and iron and folate intake. The macronutrients were expressed as the percentage of energy intake (%TEI) and the nutritional density of each micronutrient in the diet was expressed in mg or µg per 1.000 kcal [34] while an assessment of nutrient and food intake was categorized according to the processing degree as defined by the NOVA classification, which consists of dividing foods into four groups: in natura or minimally processed, culinary ingredients, processed and ultra-processed [36,37]. For the present study, the percentage of relative energy intake from UPFs were distributed into quintiles according to the contribution of UPFs to the total caloric value of the diet (% kcal), according to the cut-off points of the distribution quintiles. The assessment of iron, folate and energy consumption by pregnant women was also performed using the 24hr. Nutrients from food supplementation were not considered in the calculation of food consumption.

Data Analysis
Descriptive analysis was performed, with calculation of relative frequency distributions for categorical variables and mean and 95% confidence interval (95% CI) for continuous variables. Bivariate analysis was performed using simple linear regression considering the consumption of iron and folate (mg/1000 kcal) as the outcome variable. Multiple linear regression models were performed to predict the consumption of iron and folate (mg/1000 kcal), based on explanatory variables such as age, gestational age (categorized in trimester), family allowance (no/yes), schooling (categorized into 4 categories), meal at home (less than 3 days a week/more than 3 days a week), pre-gestational BMI (categorized), planned pregnancy (no/yes) and consumption of ultra-processed foods (categorized into quintile of total caloric value). It should be emphasized that the minimum sample size for conducting multiple linear regression is 146, considering that this study is evaluating ten independent variables and two adjusting variables [38].
For the construction of the multiple linear models, the p ≤ 0.20 value obtained in the bivariate analysis was used as criteria for the inclusion of explanatory variables. In the final models, the Backward Method was used, and those variables with lower significance (higher p-value) were removed one by one. The procedure was repeated until all variables present in the models had statistical significance (p < 0.05). The models were adjusted by the variables "ferrous sulfate supplementation" or "folic acid supplementation" (no/yes) and "use multivitamin" (no/yes). The significance of the final models were evaluated by the F test of the analysis of variance and the goodness of fit by the coefficient of determination (R 2 ). Residuals were evaluated according to normality, homoscedasticity, linearity and independence assumptions. In addition, multicollinearity was verified between the variables included in the models.
All analyzes were performed using the Stata statistical program, version 17.0 (StataCorp. College Station, TX, USA).

Ethical Aspects
The study was submitted and approved by the Research Ethics Committee of the Faculty of Health of the University of Brasília (UnB), under number 2,977,035 and by the Research Ethics Committee of the Federal District Health Department under number 3,489,243. All pregnant women signed the TCLE before starting the interview.

Results
The study included 217 pregnant women ( Figure 1) aged between 20 and 50 years. It was observed that 44.65% of the pregnant women were in the third gestational trimester. Regarding sociodemographic information, 75.6% of pregnant women declared themselves to be brown or black and 11.74% were beneficiaries of social welfare income transfer program.    Tables 2 and 3 present the variables associated with the consumption of iron (mg/1000 kcal/day) and folate (µg /1000 kcal/day) by pregnant women. Table 4 describes the final model of multiple linear regression of variables associated with iron consumption during pregnancy. The independently associated factors with iron consumption were schooling and UPFs intake. The highest quintile of consumption of UPFs was associated with lower consumption of iron. Pregnant women with high school education degree presented higher iron intake compared to those with elementary school degree. According to the standardized beta assessment, the factor that most influenced iron consumption was the consumption of UPFs. Table 5 describes the final multiple linear regression model of variables associated with folate consumption during pregnancy. The factors independently associated with folic acid consumption were schooling, gestational age, planned pregnancy, and consumption of UPFs. Pregnant women with high school education degree showed higher consumption compared to those with elementary school degree, the second trimester participants presented higher consumption of folate compared to those who were in the first trimester, and those who had a planned pregnancy also showed higher consumption of folate. The highest quintile of UPFs consumption was associated with lower consumption of folic acid. According to the standardized beta assessment, the factor that most influenced the consumption of folate was the consumption of UPFs.

Discussion
The presented data, according to the multivariate model, highlighted the association of higher UPFs consumption and low iron and folate intake in pregnant women assisted by PHC. In addition, high school degree, second gestational period and pregnancy planning were associated with a higher iron and folate intake. This is the first study to evaluate the impact of UPFs on iron and folate micronutrient content of the diet consumed by pregnant women in the capital of Brazil. The scientific community has recently focused attention on the importance of adequate nutrition during the first one thousand days of life, period from pregnancy to early childhood. The deficiency of iron and folate nutrients are related to nutritional deficiencies, anemia, and can lead to serious outcomes, such as an increased risk of infant death, such as increased birth weight and infant death [10,11].
The mean iron intake and folate of the evaluated pregnant women were below the requirement during pregnancy [39]. It is well known that the two major sources of dietetic iron are animal foods (beef, liver, offal, poultry, pork, fish and shellfish) and plant foods (dark green vegetables, beans, lentils) [40,41]. Folate is present in dark green vegetables as well as in fortified foods (in the form of folic acid) [42,43]. The pregnant women should be vigilant about their food intake to maintain the adequate levels of micronutrients. To reinforce the adequate consumption of in natura and minimally processed foods, sources of iron and folate according to Dietary Guidelines for the Brazilian Population, seems to be an important strategy to prevent micronutrients deficiency. Although, it has been observed due to the physiological requirements, that pregnant woman group will hardly be able to obtain the adequate amount of iron only through food intake, which justifies the need for supplementation at this stage [39,44].
A systematic review with studies from Ethiopia, Kenya, Nigeria and South Africa showed higher prevalence of anemia, iron deficiency, and iron deficiency anemia in pregnant women when compared to women of reproductive age, but the folate inadequacy was reduced in pregnant women due to micronutrient fortification and supplementation [45]. The ECLIPSES study, a longitudinal study conducted on 793 non-supplemented pregnant women in Spain, showed that women did not increase their energy and nutritional intake during pregnancy and postpartum and they had a high risk of iron and folate deficient intake [46]. Others studies in French-Canadian [47], Nordic population [48] and US [49] showed iron and acid folic inadequacy in pregnant women. Studies carried out in different Brazilian states showed a higher prevalence of insufficient iron and folate intake by pregnant women. In Minas Gerais State, a study with 492 pregnant women observed a prevalence of 49.2% of insufficient folate intake considering the diet with fortified foods [18] and in Rio de Janeiro State, a prevalence of 63.7% of folate deficiency was reported by 201 pregnant women who entered the study [50]. Other study carried out in São Paulo State with 30 pregnant women, also showed inadequate consumption of iron and folate [19]. A Cochrane review's meta-analyses evaluating 21 clinical trials (involving 5490 pregnant women) showed that the intermittent oral iron and folic acid supplementation may be a feasible alternative to daily iron supplementation among pregnant women populations where the prevalence of anemia is less than 20% [51]. These results taken together highlights the importance of dietary diversification, food fortification and drug supplementation as a combined strategy to assure iron and folate adequacy to the pregnant group.
In Brazil, two health public policies help to control iron deficiency anemia in pregnant women and children. First, the mandatory fortification of wheat and corn flours with iron and folic acid by food industry, which consists of the mandatory addition of 4 to 9 mg of iron and 140 to 220 µg of folic acid in wheat and corn flour per 100 g of the product [52]. Several countries in South and Central America have also instituted food fortification as a combat resource nutritional deficiency. Costa Rica, Chile, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Panama, Puerto Rico have institutionalized fortification policies [53]. Fujimori et al (2011) evaluated the retrospective data from 12,119 medical records of Brazilian pregnant women and showed a reduction in anemia prevalence (fell from 25% to 20%) after flour fortification with iron [54].
Second, the National Iron Supplementation Program (NISP), which recommends prophylactic supplementation of 40 mg of elemental iron daily throughout pregnancy and 120 mg in the case of already anemic pregnant women [15,16,41,55], whereas micronutrient supplementation are available for free to pregnant women assisted by PHC. Although the program is national, the resource is decentralized, and each municipality is responsible for purchasing and distributing the supplement. About one third of the PHC participants declare to not use iron supplementation, despite the gratuity of the program, this gap in the coverage of the NISP may be related to factors such as the unavailability of the supplement in the PHC units, the unavailability of medication or distribution without proper guidance, the non-attendance of pregnant women in medical appointments or their non-adherence to the prescription [12,56].
The total caloric value mean from ultra-processed foods by pregnant women in our study was 22.4%, and the highest quintile of consumption of UPFs was associated with lower consumption of iron and folate. The study of Graciliano et al (2021) evaluated a northeast region Brazilian pregnant women cohort and observed similar UPFs consumption (22.2%). Also, showed an association with reduced consumption of rice, beans, meat, fruits and vegetables, sources of iron and folate [27]. Ultra-processed foods are commonly high in sugar, fat, sodium, with attractive sensory characteristics, not stimulating satiety because they are low in fiber and generally obesogenic. Although most of the UPFs in Brazil contain synthetic micronutrients, the bioavailability of these nutrients is not known and, therefore, it cannot be said that they will have the same bioavailability as in natura or minimally processed foods [36,37].
Consumption of ultra-processed foods is associated with greater maternal weight gain and greater adiposity in the newborn [23,25], and with the reduction of iron intake and the quality of the pregnant woman's diet attended by PHC in Brazil [27]. Another cross-sectional study carried out in Brazil with 125 high-risk pregnant women in the third trimester of gestation showed the majority of the calories intake was from unprocessed foods (52.4%), followed by UPFs (25.5%). The fiber, calcium, folate and iron consumption was below the recommendations for pregnant women, corroborating our results [22].
The study of Crivellenti et al., aimed to develop a Diet Quality Index Adapted for Pregnant Women and showed a low ratio of women who reached the maximum score for the consumption of folate and iron, suggesting poor quality of the diet in pregnant women [57]. Consumption of UPFs was the main predictor of iron and folate intake. This result deserves to be highlighted given the importance of these nutrient for the prevention of anemia, a condition that can increase maternal and child morbidity and mortality. According to Nilson et al. (2022) approximately 57,000 premature deaths were estimated as attributable to the UPF consumption in Brazil in 2019 [58], reinforcing the impact of industrial food processing on preventable deaths. Thus, considering the increasing consumption of ultra-processed foods in the Brazilian population in general and in pregnant women, the high consumption of UPFs during pregnancy has important clinical significance given its negative impactive on maternal and neonatal health [22][23][24]59]. Interestingly, despite the influence of dietary energy density on the nutritional profile of the whole diet, there is no specific value for UPFs intake during pregnancy. The consumption of UPFs impacts the quality of the pregnant woman's diet and it is necessary to encourage the consumption of fresh and minimally processed foods at the expense of UPFs consumption by pregnant women, as recommended by the Dietary Guidelines for the Brazilian Population [37].
The pregnant women with high school degree education had higher iron and folate consumption compared to those with elementary school. Others studies conducted in Brazil also found association between low education levels, insufficient folate intakes in pregnant women and iron deficiency anemia [13,18,19]. Rodrigues et al (2015) showed an association between low folate consumption and low income, in addition to a higher prevalence in younger pregnant women and in those who had fewer meals per day [18]. Regarding the quality of the diet of pregnant women, a study carried out in China showed that limited schooling, occupation and low income were also negatively associated with adequate diet [60]. According to the United Nations Development Program -UNDP [61], the mother's education level influences on adequate prenatal care during pregnancy. Indeed, women with higher education degrees and higher-income have significantly higher chances of having at least six prenatal medical appointments and one puerperal appointment than those in a more unfavorable situation [62].
Prenatal care adherence during pregnancy is an important health factor associated with optimum outcomes. It is possible to monitor the baby's development and maternal health, directly impacting the mother's and child's health. Study shows that the late initiation of prenatal care is associated with the development of anemia during pregnancy and deserves greater attention from the public sector [12]. Another study showed a higher prevalence of anemia among women who did not undergo adequate prenatal care and pregnant women who had multiple pregnancies, showing the importance of adequate health care as a factor in the prevention of anemia and adverse gestational outcomes [63].
The pregnancy planning was associated with a higher consumption of folate. This result corroborates a study carried out in Minas Gerais State, which also associates a higher prevalence of inadequate folate intake in women who did not plan the pregnancy [18]. Our hypothesis is that pregnant women who had planned their pregnancies are more careful with the nutritional profile of diet and, therefore, have a higher consumption of folate for food. Our study did not consider folic acid supplementation in food intake analysis; therefore, our results cannot extend the supplementation interpretation. Considering that changes in quantitative and/or qualitative nutrient intake may play a crucial role in fetal programming and consequently influence the presence of non-communicable disease in life, it is worth to discuss pregnancy planning as an important tool to enhanced nutritional profile of diet and to enhance adherence to the folic acid supplement [64,65].
Regarding the gestational period, the second trimester participants presented higher consumption of folate compared to those who were in the first trimester. A study carried out with 79 pregnant women in Canada evaluated changes in diet quality according to the gestational trimester using the Canadian Healthy Eating Index (HEI). Despite not finding variation in the total score, the authors showed that the adequacy of the diet, fruit consumption and total vegetables, saturated and unsaturated fats decreased significantly throughout pregnancy [66]. This finding suggests the importance of the specialized multidisciplinary care throughout pregnancy.
Possible limitations of this study are related to the cross-sectional study design that does not allow causality analysis. The self-reported 24hr assessment depends on participant memory, cooperation, and communication abilities, and might contain some errors due to memory biases and over/underreporting. Lastly, the consumption of supplements in the food intake analysis was not considered, since there was not enough data collected. Despite these limitations, it is important to highlight the study strengths. To date, this is the first study evaluating iron and folate intake with data from Brazilian pregnant women from the Federal District region with robust methods. The dietary assessment was applied by well-trained nutrition researchers using the 5-steps multiple pass methodology. In addition, to enhance accuracy of dietary recall and to reduce bias the photographic manual of food portions was used when collecting food intake data. The results of the present study indicate important public health implications, since the higher UPFs consumption was associated with lower intake of iron and folic acid nutrients, leading to a poor nutritional profile of the diet of pregnant women assisted by PHC.

Conclusions
The consumption of ultra-processed foods and schooling were the main factors independently associated with both iron intake and folate intake in pregnant women attended in PHC. Folate consumption was also associated with the gestational period and pregnancy planning. Therefore, we suggest reinforcing the recommendations of the Dietary Guidelines for the Brazilian Population, to focus on the importance of pregnancy planning, and to reinforce the importance of early prenatal care in order to reduce adverse maternal and neonatal outcomes.